Functional ryanodine receptors in the plasma membrane of RINm5F pancreatic β-cells

Abstract

Ryanodine receptors (RyR) are Ca2+ channels that mediate Ca2+ release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca2+ entry that was independent of store-operated Ca2+ entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (γdκ = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic β-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP3 receptors (IP3R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP3R3, we showed that RyR2 mediates both the Ca2+ entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatica β-cells, where they mediate Ca2+ entry. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.